Use of phenolic antioxidants in cell bioproduction

ABSTRACT

The present invention relates to new methods and processes for culturing mammalian cells with the addition of phenolic antioxidants. Performance of the cell culturing methods and processes in their various aspects result in a higher viable cell density and higher protein titer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Pat. No.15/549,760 filed Aug. 9, 2017, now allowed, which is a 35 U.S.C. § 371National Stage patent application of International ApplicationPCT/US2016/017432, filed Feb. 11, 2016, which claims priority to U.S.Provisional Application Ser. No. 62/114,635, filed Feb. 11, 2015; theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to new methods and processes for culturingmammalian cells with the addition of antioxidants. Performance of thecell culturing methods and processes in their various aspects result ina higher viable cell density and higher protein titer.

INTRODUCTION

Current ongoing efforts to maximize bioreactor productivity in both timeand volume directly affect the scale and capital investment required fora bioreactor suite. As cells reach higher concentrations more quickly,yield is increased; therefore, the number and scale of bioreactors canbe reduced. To that end, not only cell engineering, but also culturemedia and related chemical and physical environments are used to assistcells in reaching peak performance quickly and maintaining a high levelas long as possible.

Removing or reducing reactive oxygen species (ROS) such as superoxideanions, hydrogen peroxide, hydroxyl radicals, or singlet oxygen has beenshown to improve productivity, presumably through reduction ofapoptosis. Yun et al. showed with Chinese Hamster Ovary (CHO) culturethat a combination of glutathione and iron chelators decreasedintracellular ROS levels and increased the number of viable cells(“Combined Addition of Glutathione and Iron Chelators for Decrease ofIntracellular Level of Reactive Oxygen Species and Death of ChineseHamster Ovary Cells”, J. Biosci. Bioeng., 95:124-127 (2003)). Additionof specific iron chelator combinations also reduced ROS and yieldedimproved viability. Adding several together was more effective thanadding single chelators separately. Similar results were reported uponaddition of ascorbic acid and glutathione as antioxidants to CHO cultureby Yun et al. (“Effect of Antioxidants on the Apoptosis of CHO Cells andProduction of Tissue Plasminogen Activator in Suspension Culture”, J.Biosci. Bioeng., 91:581-585 (2001)).

Antioxidants commonly used during cell culture include tocopherol,transferrin, selenium, ascorbate and reduced glutathione. Tocopherol isa membrane antioxidant with low solubility in water that functions toneutralize lipid peroxides. Transferrin is a chelator that binds ironwith such high affinity leaving no iron available to generate freeradicals. Transferrin serves as an extracellular iron transporter andstorage molecule. Glutathione is a water-soluble antioxidant tripeptide,which contains a reducing thiol group. It is ubiquitously produced inall cell types and is important for cell proliferation and viability.Selenium is a component of the antioxidant enzymes glutathioneperoxidase and thioredoxin reductase which reduce glutathione andthioredoxin, respectively. Ascorbate is a water-soluble antioxidant thatregenerates reduced tocopherol. Most commercially-available basalmediums do not contain antioxidants as they were designed for use inconjunction with serum. The serum would typically provide the requiredantioxidants.

Recombinantly produced protein products are increasingly becomingmedically and clinically important for use as therapeutics, treatmentsand prophylactics. Therefore, the development of reliable cell cultureprocesses that economically and efficiently achieve an increased viablecell density thereby resulting in increased final protein productconcentration fulfills both a desired and needed goal in the art.

SUMMARY OF THE INVENTION

The present invention provides new processes for the production ofproteins by animal or mammalian cell cultures. These new processesachieve increased viable cell density, cell viability and productivity.

The inventors have identified suitable phenolic antioxidants over arange of concentrations, in different chemically defined basal and feedmedia, in small and large scale cell culture using different transfectedcell lines that produce antibody or fusion proteins.

On aspect of the invention concerns the growth of cells in antioxidantsupplemented culture media. More specifically, the cells are Chinesehamster Ovary cells and the antioxidant is added to the basal or feedmedia, or both the basal and feed media.

Another aspect of the method of this invention concerns the selection ofphenolic antioxidant from the group consisting of apigenin, catechin,chlorogenic acid, daidzein, genistein, hesperetin, melatonin,naringenin, pelargonidin, quercetin, resveratrol, rosmarinic acid andsilibinin. More specifically, one or more antioxidant is selected fromcatechin, chlorogenic acid, pelargonidin, quercetin, resveratrol,rosmarinic acid or silibinin. More specifically, the antioxidantutilized in the method of this invention is catechin or rosmarinic acid.

Another aspect of the method of this invention concerns the amount ofantioxidant added to the cell culture. An amount of antioxidant suitablefor use in the basal and/or feed medium comprises from about 0.0625 mMto about 1 mM. More specifically, the amount of antioxidant suitable foruse in the basal media comprises the lower end of the suitable range,for example, from about 0.0625 mM to 0.25 mM. The amount of antioxidantsuitable for the feed media comprises the upper end of the suitablerange, for example, from about 0.25 mM to 1 mM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the impact of 0.0625 mM, 0.25 mM and 1 mM of differentantioxidants added to the feed media M154A1B on aCD137 viable celldensity (VCD) at day 8 (peak VCD). The basal media M17IB was notsupplemented with antioxidant. The cell density for each condition wasnormalized to the control condition with no additional antioxidants inthe feed. FIG. 2 shows the impact of 0.0625 mM, 0.25 mM and 1 mM ofdifferent antioxidants added to the feed media M154A1B on productivityof the cultured aCD137 cells. The basal media M17IB was not supplementedwith antioxidant. The final titer (harvested on days 14) for eachcondition was normalized to the control condition with no additionalantioxidants in the feed. FIG. 3A-C show the impact of catechin,chlorogenic acid, quercetin, resveratrol and rosmarinic acid on aCD137cell culture (A) viable cell density, (B) normalized protein titer, and(C) lactate concentration. The antioxidants were added to basal mediaM17IB at 0.07 mM and feed media M154A1B at 1 mM. The media were thenused in a standard shaker flask fed-batch culture with aCD137 cells.

FIG. 4A-D show the impact of catechin(□) on aCD137 cell culture (A)viable cell density, (B) cell viability, (C) normalized protein titer,and (D) ammonium concentration. Catechin was added to feed media F3.2 at1 mM. The media was then used in a standard shaker flask fed-batchculture with aCD137 cells together with basal media B1

FIG. 5A-E shows the impact of rosmarinic acid (♦) and catechin (▴) onaCD137 cell culture (A) viable cell density, (B) cell viability, (C)normalized protein titer, (D) lactate concentration and (E) ammoniumconcentration. Rosmarinic acid or catechin were added to basal mediaM17IB at 0.1 mM. The media was then used in a standard 7-liter fed-batchculture together with feed media M154A1B.

FIG. 6A-D shows the impact of rosmarinic acid (▴) on aCD137 cell culture(A) viable cell density, (B) cell viability, (C) normalized proteintiter, and (D) lactate concentration. Rosmarinic acid was added to basalmedia M17IB at 0.1 mM. The media was then used in a standard fed-batchculture together with feed media M154A1B.

FIG. 7A-D shows the impact of rosmarinic acid addition to either basalmedia B1 at 0.1 mM (RA-Basal) or feed media F3.2 at 1 mM (RA-Feed), orboth (RA-Basal+Feed) on (A) viable cell density, (B) cell viability, (C)normalized protein titer and (D) ammonium concentration. The media wasthen used in a standard shake flask with myostatin cells.

FIG. 8A-C shows the impact of rosmarinic acid (RA-Basal) or catechin(Cat-Basal) addition to basal media B1 at 0.1 mM on (A) viable celldensity, (B) cell viability, (C) normalized protein titer and (D)ammonium concentration. The media was used in a 20-liter reactor withmyostatin cells together with feed media F3.2.

FIG. 9A-C shows the impact of catechin addition to feed media 154A1 at0.34 μM, 3.45 μM, 34.45 μM or 344.52 μM on (A) viable cell density, (B)cell viability and (C) normalized protein titer. Basal media M17IBwithout catechin was used for the culture. The media was used instandard shake flasks with aCD40L cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to an enhanced process (method) for thepreparation of recombinant proteins by mammalian cell cultures,specifically CHO cell cultures, in which the viable cell density andprotein titer are increased by the use of a newly-described mediastrategy in which antioxidants are added to the cell cultures. Theantioxidants are provided to the cell cultures in an amount that iseffective for attaining and maintaining viable cell density andtherefore maximizing protein production until the end of a culturingrun. As described herein, a variety of antioxidant supplementationregimens are encompassed by the present invention.

Culturing Processes Involving Antioxidant Addition

In accordance with this invention, the addition of antioxidants to thecell culture increases and/or maintains viable cell density during theproduction run. The presence of an effective amount of antioxidants inthe basal media and/or over the course of the culturing process wasfound to result in an increased protein titer. To maintain and/orsustain the viable cell density antioxidants are added to the basal andor feed media. The processes and methods of the present invention aresuited to both small (e.g., 50 L-100 L) and large scale (e.g., 500 L andgreater) cell cultures. In addition, the methods of the presentinvention are particularly suited to cells grown and maintained asfed-batch cultures, of both small and large scale. In addition, avariety of culture media as known in the art can be used in theculturing methods of this invention.

An antioxidant is a molecule that inhibits the oxidation of othermolecules. Oxidation is a chemical reaction that transfers electrons orhydrogen from a substance to an oxidizing agent. Oxidation reactions canproduce free radicals. in turn, these radicals can start chainreactions. When the chain reaction occurs in a cell, it can cause damageor death to the cell. Antioxidants terminate these chain reactions byremoving free radical intermediates, and inhibit other oxidationreactions. They do this by being oxidized themselves, so antioxidantsare often reducing agents such as thiols, ascorbic acid, or polyphenols.

A polyphenol antioxidant is a type of antioxidant containing apolyphenolic or natural phenol substructure. Although all polyphenolshave similar chemical structures, there are some distinctivedifferences. Based on these differences polyphenols are subdivided intoseveral major subclasses including: phenolic acids (such as rosmarinicacid and chlorogenic acid, stilbens (such as resveratrol), tannins,flavonolignans (such as silibinin) and flavonoids.

Flavonoids are the largest family of polyphenolic antioxidants.Flavonoids are further divided in several subclasses including:anthocyanins (such as pelargonidin), flavanols (such as catechin),flavaones (such as naringenin and hesperetin), flavonols (such asquercetin), flavones (such as apigenin), and isoflavones (such asgenistein and daidzein).

An advantage of the present invention is that protein production costsare reduced by the increase in protein production as achieved by theculturing methods described herein. In a preferred embodiment, higherviable cell density was maintained throughout the culture run whenantioxidants were included in the basal and/or feeding medium suppliedto the cell culture as shown in FIGS. 1-9.

One embodiment of the invention involves the addition of antioxidant tothe basal media to allow for the production of large amounts of proteinindependent of the reactor scale as shown in FIGS. 5, 6, 7 and 8.

One embodiment of the invention involves the maintenance of theantioxidant feeding strategy throughout a production run, preferably,with daily feeding, to allow for the production of large amounts ofprotein independent of the reactor scale as shown in FIGS. 1, 2, 4, 7and 9.

One embodiment of the invention involves the addition of antioxidant tothe basal and feed media to allow for the production of large amounts ofprotein independent of the reactor scale as shown in FIGS. 3 and 7.

An effective concentration of antioxidants as a component in a basal orfeed media, increases, maintains and/or sustains viable cell densitythroughout the production run. The type and amount of antioxidantsuitable for use in the basal or feed medium can be determined by theskilled practitioner based on the reactor size and volume of theculture. The methods of the present invention are suitable for allreactor scales at which protein production occur, including, but notlimited to, large and small production scale, and reactor scale, e.g.,large scale cultures or commercial scale cultures, e.g., over 50 L, morepreferably over 500 L. For example, the antioxidant supplementationstrategy is applicable for production scale cultures, e.g., having avolume of about 50 liters (50 L) or less, as well as for reactor scalecultures, which can have a volume of several hundreds or thousands ofliters.

In accordance with the methods of this invention, one or moreantioxidant is selected from the group consisting of apigenin, catechin,chlorogenic acid, daidzein, genistein, hesperetin, melatonin,naringenin, pelargonidin, quercetin, resveratrol, rosmarinic acid andsilibinin; preferably, one or more antioxidant is selected fromcatechin, chlorogenic acid, pelargonidin, quercetin, resveratrol,rosmarinic acid and silibinin; more preferably, one or more antioxidantis selected from catechin, chlorogenic acid, quercetin, resveratrol androsmarinic acid; more preferably, the antioxidant is catechin orrosmarinic acid.

In accordance with the methods of this invention, basal or feed mediumconcentration of one or more of the antioxidants selected from the listabove is preferably provided in an amount which affords a highersustained or maintained viable cell density in the culture, or reactor,during the culturing process. An amount of antioxidant suitable for usein the basal and/or feed medium comprises from about 0.0625 mM to about1 mM, preferably about 0.1 mM to about 1 mM. More specifically, theamount of antioxidant suitable for use in the basal media comprises thelower end of the suitable range, for example, from about 0.0625 mM to0.25 mM, and the amount of antioxidant suitable for the feed mediacomprises the upper end of the suitable range, for example, from about0.25 mM to 1 mM.

As specific yet nonlimiting examples, 1 mM catechin in the feed mediumis suitable for use in the culturing method of the invention; or 0.1 mMrosmarinic acid or catechin in the basal media is suitable for use inthe culturing method of the invention; or 0.1 mM rosmarinic acid in thebasal media and 1 mM rosmarinic acid in the feed media is suitable foruse in the culturing method of the invention.

Cell cultures are fed with feeding medium containing antioxidants of theinvention using a variety of feeding schedules or regimens to deliverand maintain the antioxidants in the cultures in amounts that sustain aviable cell density effective antioxidant concentration therebyincreasing protein production. In general, the culturing methods of thepresent invention comprise the feeding of cell cultures with theantioxidants of the invention in the feeding medium more than one timeduring the culture run. It is to be understood that the culture volumecontributed by the feeding medium at the end of a culture run typicallycomprises approximately 30-60% of the original culture volume.

The cell cultures can be fed the antioxidants of the invention on adaily basis, or on other than a daily basis, e.g., less often than onceper day, and at varying intervals, preferably timed intervals, includingevery other day, every third day, every fourth day, and the like. Forexample, the feeding of cell cultures with the antioxidants of theinvention, preferably with antioxidant-containing feeding medium, can beperformed once per day, more than once per day, or less than once perday, and can occur one time, two times, or more than two times, e.g.,three, four, five or more times, during the total culture run. In oneembodiment, the cells are fed with antioxidants more than once.

Also encompassed by this invention is a continuous feeding schedule, forexample, involving a continuous infusion of the antioxidants of theinvention, preferably an antioxidant-containing feeding medium, into thecultures. In such a continuous feeding regimen, the cultures receiveantioxidants, preferably in feeding medium, for example, as acontinuously-supplied “drip”, or infusion, or other automated additionto the culture, in a timed, regulated, and/or programmed fashion so asto achieve and maintain the appropriate amount of antioxidant in theculture. Most preferred is a feeding regimen comprising a one time perday bolus feed with antioxidant, preferably with feeding mediumcontaining antioxidant on each day of the culture run, from thebeginning of the culture run to the day of harvesting the cells.

In accordance with the invention, the antioxidants of the invention canbe fed to the cell culture at any of the aforementioned intervals insome way other than in the feed medium. As non-limiting examples, theantioxidants can be fed to the culture in DMSO, ethanol or water. As anon-limiting example, the culture may be fed with antioxidants and alsofed with a feed medium, i.e., there may be more than one compositionbeing fed.

As used herein, the term “feed” refers to any addition of any substancemade to a culture after inoculation. Feeding can be one or moreadditions.

As used herein, the term “inoculation” refers to the addition of cellsto starting medium to begin the culture.

As used herein, the terms “feed medium”, “feed media” and “feedingmedium” refer to a medium containing one or more nutrients that is addedto the culture beginning at some time after inoculation.

As used herein, the term “basal medium” and “basal media” refers tostarting medium to which cells are added to begin the culture.

In another of its embodiments, the present invention encompasses a cellculture method or process of increasing protein titer in a cell culture,comprising the addition of catechin to the culturing medium. A relatedembodiment involves a cell culture method in which catechin is suppliedto the culture for the duration of the production run, and preferably ispresent in the feeding medium. A preferred embodiment relates toprocesses of culturing cells comprising a daily feeding of the culturewith medium containing catechin. The addition of catechin to the cellsin culture as provided by this method, preferably supplied more than onetime during the culture run, more preferably, on a daily basis,increases, maintains viable cell density in the culture, allowing thecells to continue to produce protein, thereby maximizing proteinproduction. Another related embodiment involves a cell culture method inwhich catechin is supplied to the culture in the basal media. Theaddition of catechin to the cells in the basal media as provided by thismethod increases and/or maintains viable cell density in the culture,allowing the cells to continue to produce protein, thereby maximizingprotein production. Another related embodiment involves a cell culturemethod in which catechin is supplied to the culture in the basal mediaas well as for the duration of the production run, preferably in thefeeding medium. A preferred embodiment relates to processes of culturingcells comprising cell inoculation in an antioxidant supplemented basalmedia and a daily feeding of the culture with medium containingcatechin. The addition of catechin to the cells in culture as providedby this method increases and/or maintains viable cell density in theculture, allowing the cells to continue to produce protein, therebymaximizing protein production

In another of its embodiments, the present invention encompasses a cellculture method or process of increasing protein titer in a cell culture,comprising the addition of rosmarinic acid to the culturing medium. Arelated embodiment involves a cell culture method in which rosmarinicacid is supplied to the culture for the duration of the production run,and preferably is present in the feeding medium. A preferred embodimentrelates to processes of culturing cells comprising a daily feeding ofthe culture with medium containing rosmarinic acid. The addition ofrosmarinic acid to the cells in culture as provided by this method,preferably supplied more than one time during the culture run, morepreferably, on a daily basis, increases, maintains viable cell densityin the culture, allowing the cells to continue to produce protein,thereby maximizing protein production. Another related embodimentinvolves a cell culture method in which rosmarinic acid is supplied tothe culture in the basal media. The addition of rosmarinic acid to thecells in basal media as provided by this method increases and/ormaintains viable cell density in the culture, allowing the cells tocontinue to produce protein, thereby maximizing protein production.Another related embodiment involves a cell culture method in whichrosmarinic acid is supplied to the culture in the basal media as well asfor the duration of the production run, preferably in the feedingmedium. A preferred embodiment relates to processes of culturing cellscomprising cell inoculation in an antioxidant supplemented basal mediaand a daily feeding of the culture with medium containing rosmarinicacid. The addition of rosmarinic acid to the cells in culture asprovided by this method increases and/or maintains viable cell densityin the culture, allowing the cells to continue to produce protein,thereby maximizing protein production.

In accordance with this invention, viable cell density is increasedabout 10-100% when the cell culture is supplemented with antioxidants,as compared with cell cultures in the absence of antioxidantsupplementation.

In accordance with this invention, protein titer is increased, onaverage, about 30-130% when cell cultures are supplemented withantioxidants, as compared with cell cultures in the absence ofantioxidant supplementation.

Screening Natural Phenolic Antioxidants For Bioproduction

Following the cell culture process described in Example 1, thirteennovel antioxidants (Table 1) were added to the feed media utilized inaCD137 cell culture. Three antioxidant concentrations were evaluated: 1mM (Run #1-13), 0.25 mM (Run #17-29), and 0.0625 mM (Run #33-45). Theantioxidants were dissolved in either pure ethanol or dimethyl sulfoxide(DMSO) at 50 mM stock concentration and added to feed media M154A1B atthe designed final concentrations. The corresponding solvent negativecontrols were run number 16, 32, and 48. Two traditional antioxidants,ascorbic acid (Run #14, 30, and 46) and reduced glutathione (Run #15,31, and 47), served as positive controls. The basal media M17IB was notsupplemented with antioxidant.

ABLE 1 List of Antioxidants and Concentrations Evaluated ConcentrationRun# Component (mM) 1 Catechin 1 2 Apigenin 1 3 Naringenin 1 4Hesperetin 1 5 Quercetin 1 6 Pelargonidin 1 7 Genistein 1 8 Daidzein 1 9Resveratrol 1 10 Melatonin 1 11 Chlorogenic acid 1 12 Rosmarinic acid 113 Silibinin 1 14 Ascorbic Acid 1 15 L-Glutathione reduced 1 16Solvent-DMSO 1% v/v 17 Catechin 0.25 18 Apigenin 0.25 19 Naringenin 0.2520 Hesperetin 0.25 21 Quercetin 0.25 22 Pelargonidin 0.25 23 Genistein0.25 24 Daidzein 0.25 25 Resveratrol 0.25 26 Melatonin 0.25 27Chlorogenic acid 0.25 28 Rosmarinic acid 0.25 29 Silibinin 0.25 30Ascorbic Acid 0.25 31 L-Glutathione reduced 0.25 32 Solvent-Ethanol 1%v/v 33 Catechin 0.0625 34 Apigenin 0.0625 35 Naringenin 0.0625 36Hesperetin 0.0625 37 Quercetin 0.0625 38 Pelargonidin 0.0625 39Genistein 0.0625 40 Daidzein 0.0625 41 Resveratrol 0.0625 42 Melatonin0.0625 43 Chlorogenic acid 0.0625 44 Rosmarinic acid 0.0625 45 Silibinin0.0625 46 Ascorbic Acid 0.0625 47 L-Glutathione reduced 0.0625 48Solvent-H₂O N/A

As shown in FIG. 1, addition of the antioxidants to the feed mediaaffected viable cell density (VCD) on day 8 (peak VCD). In someinstances, viable cell density was increased by as much as 30% by theaddition of antioxidant compared to the controls (DMSO, ethanol, orwater).

As shown in FIG. 2, addition of the antioxidants to the feed media alsoaffected the productivity of the cultured cells. In some instances,protein titer was increased by as much as 80% by addition of antioxidantcompared to the controls (DMSO, ethanol, or water).

The antioxidants with the greatest positive impact on protein titer arepresented in Table 2 below. Addition of catechin, chlorogenic acid,resveratrol, and rosmarinic acid increased protein titer when feed atall three concentrations (0.0625-1 mM). Quercetin feed at 0.25 mM and 1mM increased protein titer. Pelargonidin and silibinin feed increasedprotein titer when feed at 1 mM.

TABLE 2 List of Antioxidants and Concentrations that Improved ProteinTiter Component VCD % Titer % Concentration (mM) Catechin 102.01 116.671 Catechin 93.52 112.70 0.25 Catechin 98.66 111.90 0.0625 Chlorogenicacid 129.57 151.59 1 Chlorogenic acid 109.35 109.92 0.25 Chlorogenicacid 115.85 110.71 0.0625 Pelargonidin 104.47 118.25 1 Quercetin 123.11128.97 1 Quercetin 119.28 111.90 0.25 Resveratrol 107.48 144.05 1Resveratrol 92.24 111.90 0.25 Resveratrol 102.53 112.70 0.0625Rosmarinic acid 124.08 180.16 1 Rosmarinic acid 125.43 146.83 0.25Rosmarinic acid 129.25 132.14 0.0625 Silibinin 99.11 144.44 1

Cells, Proteins and Cell Culture

In the cell culture processes or methods of this invention, the cellscan be maintained in a variety of cell culture media, i.e., basalculture media, as conventionally known in the art. For example, themethods are applicable for use with large volumes of cells maintained incell culture medium, which can be supplemented with nutrients and thelike. Typically, “cell culturing medium” (also called “culture medium”)is a term that is understood by the practitioner in the art and is knownto refer to a nutrient solution in which cells, preferably animal ormammalian cells, are grown and which generally provides at least one ormore components from the following: an energy source (usually in theform of a carbohydrate such as glucose); all essential amino acids, andgenerally the twenty basic amino acids, plus cysteine; vitamins and/orother organic compounds typically required at low concentrations; lipidsor free fatty acids, e.g., linoleic acid; and trace elements, e.g.,inorganic compounds or naturally occurring elements that are typicallyrequired at very low concentrations, usually in the micromolar range.Cell culture medium can also be supplemented to contain a variety ofoptional components, such as hormones and other growth factors, e.g.,insulin, transferrin, epidermal growth factor, serum, and the like;salts, e.g., calcium, magnesium and phosphate, and buffers, e.g., HEPES;nucleosides and bases, e.g., adenosine, thymidine, hypoxanthine; andprotein and tissue hydrolysates, e.g., hydrolyzed animal protein(peptone or peptone mixtures, which can be obtained from animalbyproducts, purified gelatin or plant material); antibiotics, e.g.,gentamycin; and cell protective agents, e.g., a PLURONIC® polyol(PLURONIC® F68). Preferred is a cell nutrition medium that is serum-freeand free of products or ingredients of animal origin.

As is appreciated by the practitioner, animal or mammalian cells arecultured in a medium suitable for the particular cells being culturedand which can be determined by the person of skill in the art withoutundue experimentation. Commercially available media can be utilized andinclude, for example, Minimal Essential Medium (MEM, Sigma, St. Louis,Mo.); Ham's F10 Medium (Sigma); Dulbecco's Modified Eagles Medium (DMEM,Sigma); RPMI-1640 Medium (Sigma); HYCLONE® cell culture medium (HyClone,Logan, Utah); and chemically-defined (CD) media, which are formulatedfor particular cell types. To the foregoing, exemplary media can beadded the above-described supplementary components or ingredients,including optional components, in appropriate concentrations or amounts,as necessary or desired, and as would be known and practiced by thosehaving in the art using routine skill.

In addition, cell culture conditions suitable for the methods of thepresent invention are those that are typically employed and known forbatch, fed-batch, or continuous culturing of cells, with attention paidto pH (e.g., about 6.5 to about 7.5), dissolved oxygen (O₂) (e.g.,between about 5-90% of air saturation), carbon dioxide (CO₂) (e.g.,between about 10-150%), agitation (between about 50 to 200 rpm) andhumidity, in addition to temperature (between about 30° C. to 37° C.).As an illustrative, yet nonlimiting, example, a suitable cell culturingmedium for the fed-batch processes of the present invention comprises achemically defined basal and feed medium, preferably one or bothcontaining the antioxidants of the invention (e.g., Example 1).

Animal cells, mammalian cells, cultured cells, animal or mammalian hostcells, host cells, recombinant cells, recombinant host cells, and thelike, are all terms for the cells that can be cultured according to theprocesses of this invention. Such cells are typically cell linesobtained or derived from mammals and are able to grow and survive whenplaced in either monolayer culture or suspension culture in mediumcontaining appropriate nutrients and/or growth factors. Growth factorsand nutrients that are necessary for the growth and maintenance ofparticular cell cultures are able to be readily determined empiricallyby those having skill in the pertinent art, such as is described, forexample, by Barnes et al. (Cell, 22:649 (1980)); in Mather, J. P., ed.,Mammalian Cell Culture, Plenum Press, NY (1984); and in U.S. Pat. No.5,721,121.

Numerous types of cells can be cultured according to the methods of thepresent invention. The cells are typically animal or mammalian cellsthat can express and secrete, or that can be molecularly engineered toexpress and secrete, large quantities of a particular protein into theculture medium. It will be understood that the protein produced by ahost cell can be endogenous or homologous to the host cell.Alternatively, and preferably, the protein is heterologous, i.e.,foreign, to the host cell, for example, a human protein produced andsecreted by a Chinese hamster ovary (CHO) host cell.

Examples of mammalian proteins that can be advantageously produced bythe methods of this invention include, without limitation, cytokines,cytokine receptors, growth factors (e.g., EGF, HER-2, FGF-α, FGF-β,TGF-α, TGF-β, PDGF. IGF-1, IGF-2, NGF, NGF-β); growth factor receptors,including fusion or chimeric proteins. Other nonlimiting examplesinclude growth hormones (e.g., human growth hormone, bovine growthhormone); insulin (e.g., insulin A chain and insulin B chain),proinsulin; erythropoietin (EPO); colony stimulating factors (e.g.,G-CSF, GM-CSF, M-CSF); interleukins (e.g., IL-1 through IL-12); vascularendothelial growth factor (VEGF) and its receptor (VEGF-R); interferons(e.g., IFN-α, β, or γ); tumor necrosis factor (e.g., TNF-α and TNF-β)and their receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin;brain natriuretic peptide (BNP); clotting factors (e.g., Factor VIII,Factor IX, von Willebrands factor, and the like); anti-clotting factors;tissue plasminogen activator (TPA), e.g., urokinase or human urine ortissue type TPA; follicle stimulating hormone (FSH); luteinizing hormone(LH); calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.);CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bonemorphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.);neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF);neurotrophins, e.g., 3-6; renin; rheumatoid factor; RANTES; albumin;relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viralproteins or antigens; surface membrane proteins; ion channel proteins;enzymes; regulatory proteins; antibodies; immunomodulatory proteins,(e.g., HLA, MHC, the B7 family); homing receptors; transport proteins;superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs);neuromodulatory proteins; Alzheimer's Disease associated proteins andpeptides, (e.g., A-beta), and others as known in the art. Fusionproteins and polypeptides, chimeric proteins and polypeptides, as wellas fragments or portions, or mutants, variants, or analogs of any of theaforementioned proteins and polypeptides are also included among thesuitable proteins, polypeptides and peptides that can be produced by themethods of the present invention.

Nonlimiting examples of animal or mammalian host cells suitable forharboring, expressing, and producing proteins for subsequent isolationand/or purification include Chinese hamster ovary cells (CHO), such asCHO-K1 (ATCC® CCL-61), DG44 (Chasin et al., Som. Cell Molec. Genet.,12:555-556 (1986); Kolkekar et al., Biochemistry, 36:10901-10909 (1997);and WO 01/92337 A2), dihydrofolate reductase negative CHO cells(CHO/-DHFR, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980)),and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cellstransformed by SV40 (COS cells, COS-7, ATCC® CRL-1651); human embryonickidney cells (e.g., 293 cells, or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen. Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC® CCL-10); monkey kidney cells (CV1,ATCC® CCL-70); African green monkey kidney cells (VERO-76, ATCC®CRL-1587; VERO, ATCC® CCL-81); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); human cervical carcinoma cells (HELA, ATCC®CCL-2); canine kidney cells (MDCK, ATCC® CCL-34); human lung cells(W138, ATCC® CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC® CCL-51); buffalo rat liver cells(BRL 3A, ATCC® CRL-1442); TRI cells (Mather, Annals NY Acad. Sci.,383:44-68 (1982)); MCR 5 cells; FS4 cells. Preferred are CHO cells,particularly, CHO/-DHFR cells.

The cells suitable for culturing in the methods and processes of thepresent invention can contain introduced (e.g., via transformation,transfection, infection, or injection) expression vectors (constructs),such as plasmids and the like, that harbor coding sequences, or portionsthereof, encoding the proteins for expression and production in theculturing process. Such expression vectors contain the necessaryelements for the transcription and translation of the inserted codingsequence. Methods which are well known to and practiced by those skilledin the art can be used to construct expression vectors containingsequences encoding the produced proteins and polypeptides, as well asthe appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. (1989) and in Ausubel,F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y. (1989).

Control elements, or regulatory sequences, are those non-translatedregions of the vector (e.g., enhancers, promoters, 5′ and 3′untranslated regions) that interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and host cellutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferred. The constructs for use in protein expressionsystems are designed to contain at least one promoter, an enhancersequence (optional, for mammalian expression systems), and othersequences as necessary or required for proper transcription andregulation of gene expression (e.g., transcriptional initiation andtermination sequences, origin of replication sites, polyadenylationsequences, e.g., the Bovine Growth Hormone (BGH) poly A sequence).

As will be appreciated by those skilled in the art, the selection of theappropriate vector, components for proper transcription, expression, andisolation of proteins produced in eukaryotic expression systems is knownand routinely determined and practiced by those having skill in the art.The expression of proteins by the cells cultured in accordance with themethods of this invention can be placed under the control of promoterssuch as viral promoters, e.g., cytomegalovirus (CMV), Rous sarcoma virus(RSV), phosphoglycerol kinase (PGK), thymidine kinase (TK), or theα-ACTIN® promoter. Further, regulated promoters confer inducibility byparticular compounds or molecules, e.g., the glucocorticoid responseelement (GRE) of mouse mammary tumor virus (MMTV) is induced byglucocorticoids (Chandler, V. et al., Cell, 33:489-499 (1983)). Also,tissue-specific promoters or regulatory elements can be used (Swift, G.et al., Cell, 38:639-646 (1984)), if necessary or desired.

Expression constructs can be introduced into cells by a variety of genetransfer methods known to those skilled in the art, for example,conventional gene transfection methods, such as calcium phosphateco-precipitation, liposomal transfection, microinjection,electroporation, and infection or viral transduction. The choice of themethod is within the competence of the skilled practitioner in the art.It will be apparent to those skilled in the art that one or moreconstructs carrying DNA sequences for expression in cells can betransfected into the cells such that expression products aresubsequently produced in and/or obtained from the cells.

In a particular aspect, mammalian expression systems containingappropriate control and regulatory sequences are preferred for use inprotein expressing mammalian cells of the present invention. Commonlyused eukaryotic control sequences for generating mammalian expressionvectors include promoters and control sequences compatible withmammalian cells such as, for example, the cytomegalovirus (CMV) promoter(CDM8 vector) and avian sarcoma virus (ASV) πLN vector. Other commonlyused promoters include the early and late promoters from Simian Virus 40(SV40) (Fiers et al., Nature, 273:113 (1973)), or other viral promoterssuch as those derived from polyoma, Adenovirus 2, and bovine papillomavirus. An inducible promoter, such as hMTII (Karin et al., Nature,299:797-802 (1982)) can also be used.

Examples of expression vectors suitable for eukaryotic host cellsinclude, but are not limited to, vectors for mammalian host cells (e.g.,BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2,pRc/RSV, pSFV1 (Life Technologies); pVPakc Vectors, pCMV vectors, pSG5vectors (Stratagene), retroviral vectors (e.g., pFB vectors(Stratagene)), pcDNA-3 (Invitrogen), adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)), or modified forms of any of theforegoing. Vectors can also contain enhancer sequences upstream ordownstream of promoter region sequences for optimizing gene expression.

A selectable marker can also be used in a recombinant vector (e.g., aplasmid) to confer resistance to the cells harboring (preferably, havingstably integrated) the vector to allow their selection in appropriateselection medium. A number of selection systems can be used, includingbut not limited to, the Herpes Simplex Virus thymidine kinase (HSV TK),(Wigler et al., Cell, 11:223 (1977)), hypoxanthine-guaninephosphoribosyltransferase (HGPRT), (Szybalska et al., Proc. Natl. Acad.Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy etal., Cell, 22:817 (1980)) genes, which can be employed in tk-, hgprt-,or aprt-cells (APRT), respectively.

Anti-metabolite resistance can also be used as the basis of selectionfor the following nonlimiting examples of marker genes: dhfr, whichconfers resistance to methotrexate (Wigler et al., Proc. Natl. Acad.Sci. USA, 77:357 (1980); and O'Hare et al., Proc. Natl. Acad. Sci. USA,78:1527 (1981)); gpt, which confers resistance to mycophenolic acid(Mulligan et al., Proc. Natl. Acad. Sci. USA, 78:2072 (1981)); neo,which confers resistance to the aminoglycoside G418 (Clin. Pharmacy,12:488-505; Wu et al., Biotherapy, 3:87-95 (1991); Tolstoshev, Ann. Rev.Pharmacol. Toxicol., 32:573-596 (1993); Mulligan, Science, 260:926-932(1993); Anderson, Ann. Rev. Biochem., 62:191-21 (1993); TIB TECH,11(5):155-215 (May 1993); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology can be routinely appliedto select the desired recombinant cell clones, and such methods aredescribed, for example, in Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); inDracopoli et al., eds., Chapters 12 and 13, Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol., 150:1 (1981), which are incorporated by reference herein in theirentireties.

In addition, the expression levels of an expressed protein molecule canbe increased by vector amplification (for a review, see Bebbington etal., Chapter 8: “The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning”, DNACloning: A Practical Approach, Vol. 3, pp. 163-188, IRL Press Limited(1987)). When a marker in the vector system expressing a protein isamplifiable, an increase in the level of inhibitor present in the hostcell culture will increase the number of copies of the marker gene.Since the amplified region is associated with the protein-encoding gene,production of the protein will concomitantly increase (Crouse et al.,Mol. Cell. Biol., 3:257 (1983)).

Vectors which harbor glutamine synthase (GS) or dihydrofolate reductase(DHFR) encoding nucleic acid as the selectable markers can be amplifiedin the presence of the drugs methionine sulphoximine or methotrexate,respectively. An advantage of glutamine synthase based vectors is theavailability of cell lines (e.g., the murine myeloma cell line, NSO)which are glutamine synthase negative. Glutamine synthase expressionsystems can also function in glutamine synthase expressing cells (e.g.,CHO cells) by providing additional inhibitor to prevent the functioningof the endogenous gene.

Vectors that express DHFR as the selectable marker include, but are notlimited to, the pSV2-dhfr plasmid (Subramani et al., Mol. Cell. Biol.,1:854 (1981). Vectors that express glutamine synthase as the selectablemarker include, but are not limited to, the pEE6 expression vectordescribed in Stephens et al., Nucl. Acids. Res., 17:7110 (1989). Aglutamine synthase expression system and components thereof are detailedin PCT publications: WO 87/04462; WO 86/05807; WO 89/01036; WO 89/10404;and WO 91/06657 which are incorporated by reference herein in theirentireties. In addition, glutamine synthase expression vectors that canbe used in accordance with the present invention are commerciallyavailable from suppliers, including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.).

Types of Cell Cultures

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,continuous culture, batch culture and fed-batch culture. In a continuousculture, for example, fresh culture medium supplement (i.e., feedingmedium) is provided to the cells during the culturing period, while oldculture medium is removed daily and the product is harvested, forexample, daily or continuously. In continuous culture, feeding mediumcan be added daily and can be added continuously, i.e., as a drip orinfusion. For continuous culturing, the cells can remain in culture aslong as is desired, so long as the cells remain alive and theenvironmental and culturing conditions are maintained.

In batch culture, cells are initially cultured in medium and this mediumis neither removed, replaced, nor supplemented, i.e., the cells are not“fed” with new medium, during or before the end of the culturing run.The desired product is harvested at the end of the culturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run, i.e., the cells are“fed” with new medium (“feeding medium”) during the culturing period.Fed-batch cultures can include the various feeding regimens and times asdescribed above, for example, daily, every other day, every two days,etc., more than once per day, or less than once per day, and so on.Further, fed-batch cultures can be fed continuously with feeding medium.The desired product is then harvested at the end of theculturing/production run.

Phases of Cell Culture and Associated Parameters

The term “inoculation” refers to the addition of cells to startingmedium to begin the culture.

The growth phase of a culture is the phase during which the viable celldensity at any time point is higher than at any previous time point.

The stationary phase of a culture is the phase during which the viablecell density is approximately constant (i.e., within measuring error)over a time period of any length.

The death phase of a culture is the phase that comes after the growthphase or after the growth phase and the stationary phase, and duringwhich the viable cell density at any time point is lower than at anyprevious time point during that phase.

In a growth-associated culture process, the production phase may startduring the extended growth phase.

In a non-growth associated culture process, the production phase of cellculture may be the stationary phase.

Preferably, the culture medium is supplemented (“fed”) during theproduction phase to support continued protein production, particularlyin an extended production phase, and to attain ample quantities ofprotein product. Feeding can occur on a daily basis, or according toother schedules to support cell viability and protein production.

Antioxidants Improved Process Performance For Multiple CHO Clones aCD137Cells

In another embodiment, the dhfr-negative Chinese Hamster Ovary (CHO)cell line DG44 (Invitrogen Corp. Carlsbad, Calif.) were transfected inorder to establish a stable cell line expressing a human IgG4 antibody.

The transfected CHO DG44 cells expressing the antibody was grown in thepresence of antioxidants according to the methods of the invention.

As shown in FIG. 3, catechin, chlorogenic acid, quercetin, resveratrolor rosmarinic acid increased viable cell density and protein titer whenadded to both the basal media at 0.07 mM and the feed media at 1 mM.

Additionally, lactate production was reduced. Lactate produced in theglucose metabolism process is a major inhibitory waste product inmammalian cell cultures. High lactate concentrations may be anindication of malfunction of cell energy metabolism. Additionally, highconcentrations of lactate may also alter the pH of the cell culture.Therefore, it is preferred to maintain a lower lactate level (<5 g/L)during the course of production.

As shown in FIG. 4, catechin addition to the cell culture maintainedviable cell density and increased protein titer when added to the feedmedia at 1 mM.

Further, ammonium formation was reduced. Like lactate, ammonium is amajor inhibitory waste product in mammalian cell cultures. High ammoniumconcentrations may alter the pH of the cell culture. Additionally, it isbelieved that high ammonium level may alter the quality attributes ofthe glycoproteins produced (N-link, etc.). Therefore, it is preferred tomaintain a lower ammonium level for a cell culture process (<10 mM; <5mM mostly preferred).

As shown in FIG. 5, rosmarinic acid or catechin addition to the cellculture increased viable cell density and protein titer; and reducedammonium and lactate formation. Rosmarinic acid or catechin was added tobasal media at 0.1 mM.

As shown in FIG. 6, the positive rosmarinic effect is maintained whenthe cell culture is scaled up to 500-liter production run. Addingrosmarinic acid to the basal media at 0.1 mM increased viable celldensity and protein titer, and reduced lactate formation.

Myostatin Cells

In another embodiment, the dhfr-negative Chinese Hamster Ovary (CHO)cell line DG44 (Invitrogen Corp. Carlsbad, Calif.) was transfected inorder to establish a stable cell line expressing a recombinant humanfusion protein.

The transfected CHO DG44 cells expressing the fusion protein was grownin the presence of antioxidants according to the methods of theinvention.

As shown in FIG. 7, rosmarinic acid addition to the cell culture eitherin the basal media at 0.1 mM or feed media at 1 mM or both, the feed andbasal media, increased the viable cell density and protein titer, andreduced ammonium formation.

FIG. 8 shows that when the cell culture was scaled up to a 20-literbioreactor, the positive rosmarinic acid and catechin effect on proteintiter was maintained while the viable cell density was not changed bythe addition of rosmarinic acid or catechin to the basal media at 0.1mM.

CD40L Cells

In another embodiment, the dhfr-negative Chinese Hamster Ovary (CHO)cell line DG44 (Invitrogen Corp. Carlsbad, Calif.) was transfected inorder to establish a stable cell line expressing a recombinant humanfusion protein.

The transfected CHO DG44 cells expressing the fusion protein was grownin the presence of antioxidants according to the methods of theinvention.

As shown in FIG. 9, the increase in viable cell density and proteintiter corresponds to an increase in catechin concentration in the feedmedia (0.34 μM, 3.45 μM, 34.45 μM and 344.52 μM).

EXAMPLE 1 Cell Culture

Cells were cultured either in 50 ml spin tubes with an initial volume of25 ml and a shaking speed of 300 rpm; or 250 ml shake flasks with aninitial volume of 80˜100 ml and a shaking speed of 150 rpm on an orbitalshaker with 25 mm throw distance. Temperature for the culture wascontrolled at constant 37° C. from the beginning of the culture and wasshifted to 34° C. when viable cell density reached 10⁶ cells/ml (usuallyon day 6) and CO₂ was controlled at 6%.

A standard fed-batch culture process involved culturing the cells for 14days with feeding beginning on day 3 at a feeding volume of 3.64% ofinitial culture volume. All the media used for cultures were chemicallydefined. Chemically defined feed media M154A1B is an enriched version offeed media F3.2. While the ingredient lists are the same between the twofeed media, the concentration of a few of the ingredients is increased.Likewise, chemically defined basal media M17IB is an enriched version ofbasal media B1. For cultures in 7-liter or 500-liter reactors, dissolvedoxygen (DO) was maintained at 50% and pH was controlled between 6.8 and7.4.

Analyses

Viable cell density (VCD) and cell viability were measured off-lineusing a CEDEX® automated cell counter (Innovatis AG). Culture sampleswere also analyzed off-line using a BIOPROFILE® 400 Analyzer to monitorpH, pCO₂, pO₂, glucose, glutamine, glutamate, lactate, and ammonium(Nova Biomedical Corporation). A Protein A HPLC method was used tomeasure protein titer, which were reported as normalized values.

We claim:
 1. A cell culture process for the production of protein,comprising: a) culturing host cells which produce a protein of interestin cell culture under conditions that allow for protein production; andb) adding one or more antioxidant selected from the group consisting ofapigenin, catechin, chlorogenic acid, daidzein, genistein, hesperetin,melatonin, naringenin, pelargonidin, quercetin, resveratrol, rosmarinicacid and silibinin to the cell culture.
 2. The process according toclaim 1, wherein the antioxidant is selected from the group consistingof catechin, chlorogenic acid, pelargonidin, quercetin, resveratrol,rosmarinic acid and silibinin.
 3. The process according to claim 1,wherein the antioxidant is selected from the group consisting ofcatechin, chlorogenic acid, quercetin, resveratrol and rosmarinic acid.4. The process according to claim 1, wherein the antioxidant is catechinor rosmarinic acid.
 5. The process according to claim 1, wherein theantioxidant is added to the basal media, feed media or both.
 6. Theprocess according to claim 5, wherein the antioxidant is added to thebasal media.
 7. The process according to claim 5, wherein theantioxidant is added to the feed media.
 8. The process according toclaim 5, wherein the antioxidant is added to the both the basal and feedmedia.
 9. The process according to claim 7, wherein the feed media isadded to the cell culture on a daily basis.
 10. The process according toclaim 1, wherein the antioxidant is added to the cells in an amount ofabout 0.0625 mM to 1 mM.
 11. The process according to claim 10, whereinantioxidant is added to the cells in an amount of about 0.1 mM to 1 mM.12. The process according to claim 1, wherein the cells are mammaliancells.
 13. The process according to claim 12, wherein the mammaliancells are CHO cells.
 14. The process according to claim 1, whereinviable cell density is increased or maintained compared to the same cellculture without antioxidant supplementation.
 15. The process accordingto claim 1, wherein protein titer is increased compared to the same cellculture without antioxidant supplementation.
 16. A cell culture processfor the production of protein, comprising: a) culturing CHO cells whichproduce a protein of interest in cell culture under conditions thatallow for protein production; and b) feeding the cell culture with 1 mMrosmarinic acid added to the feed media.
 17. A cell culture process forthe production of protein, comprising culturing CHO cells which producea protein of interest in cell culture under conditions that allow forprotein production, wherein 0.1 mM rosmarinic acid is added to the basalmedia.